Process for the production of SSC's via expandase activity on penicillin G
An overall process for the preparation and recovery of 7-aminodesacetoxycephalosporanic acid (7-ADCA) via enzymatic ring expansion activity on penicillin G, using a Penicillium chrysogenum transformant strain expressing modified expandase enzyme.
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Claims
1. A modified pencillin expandase gene encoding a mutant pencillin expandase having an altered substrate specificity as compared to the corresponding wild-type pencillin expandase said modification comprising a substitution at one or more selected sites corresponding to a residue position in penicillin expandase of Streptomyces clavuligerus selected from the group consisting of arginine 74, cysteine 155, proline 157, leucine 159, phenylalanine 264, isoleucine 298, tyrosine 302, arginine 306 and arginine 266.
2. The modified pencillin expandase gene of claim 1 comprising one or more mutations selected from the group consisting of R74F; C155L; P157F; P157V; P157G; P157A; and R266N.
3. An expression system which comprises the modified pencillin expandase gene of claim 1 operably linked to control sequences for the expression of said gene.
4. Host cells modified to contain the expression system of claim 3.
5. The host cells of claim 4 which are of a microorganism.
6. The microorganism host cells of claim 5 which are cells of Penicillium chrysogenum.
7. A method to produce a modified pencillin expandase which method comprises culturing the cells of claim 4 under conditions wherein said gene is expressed to produce said pencillin expandase.
8. The method of claim 7 which further comprises recovering said pencillin expandase from said culture.
9. A method for preparing 7-aminodesacetoxycephalosporanic acid (7-ADCA) which method comprises treating penicillin G with the culture of claim 7 to produce the intermediate phenylacetyl 7-ADCA.
10. A method for preparing 7-aminodesacetoxycephalosporanic acid (7-ADCA) which method comprises treating penicillin G with the recovered pencillin expandase of claim 8 to produce the intermediate phenylacetyl 7-ADCA.
11. The process of claim 9 which further includes hydrolyzing the phenylacetyl-7-ADCA produced.
12. The process of claim 10 which further includes hydrolyzing the phenylacetyl-7-ADCA produced.
13. An improved process for the preparation and recovery of 7-aminodesacetoxycephalosporanic acid (7-ADCA) by:
- a) transforming a Penicillium chrysogenum strain with a modified pencillin expandase gene as defined in claim 1 under the transcriptional and translational regulation of fungal expression signals;
- b) fermenting said strain in a culture medium and adding to said culture medium phenylacetic acid or a salt or ester thereof suitable to yield penicillin G, which is expanded to form phenylacetyl-7-ADCA;
- c) recovering the phenylacetyl-7-ADCA from the fermentation broth;
- d) deacylating phenylacetyl-7-ADCA; and
- e) recovering the crystalline 7-ADCA.
14. The process of claim 13 wherein step (e) is a filtration step.
15. The process of claim 13, wherein step (c) is a filtration step, and by extracting the broth filtrate with an organic solvent immiscible with water at a pH of lower than about 4.5 and back-extracting the same with water at a pH between 4 and 10.
16. The process of claim 13 wherein the pencillin expandase gene is derived from Streptomyces clavuligerus or Norcardia lactamdurans.
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Type: Grant
Filed: Nov 27, 1996
Date of Patent: Jul 6, 1999
Assignee: Isis Innovation Limited (Oxford)
Inventors: John David Sutherland (Osney Island), Roelof Ary Lans Bovenberg (Rotterdam), Jan Metske van der Laan (Breda)
Primary Examiner: Robert A. Wax
Assistant Examiner: William W. Moore
Law Firm: Morrison & Foerster, LLP
Application Number: 8/757,443
International Classification: C12P 3310; C12N 1552; C12N 900; C12N 121;
